The Dynamics of Flower Development in Castanea sativa Mill.

Abstract

The sweet chestnut tree (Castanea sativa Mill.) is one of the most significant Mediterranean tree species, being an important natural resource for the wood and fruit industries. It is a monoecious species, presenting unisexual male catkins and bisexual catkins, with the latter having distinct male and female flowers. Despite the importance of the sweet chestnut tree, little is known regarding the molecular mechanisms involved in the determination of sexual organ identity. Thus, the study of how the different flowers of C. sativa develop is fundamental to understand the reproductive success of this species and the impact of flower phenology on its productivity. In this study, a C. sativa de novo transcriptome was assembled and the homologous genes to those of the ABCDE model for floral organ identity were identified. Expression analysis showed that the C. sativa B- and C-class genes are differentially expressed in the male flowers and female flowers. Yeast two-hybrid analysis also suggested that changes in the canonical ABCDE protein–protein interactions may underlie the mechanisms necessary to the development of separate male and female flowers, as reported for the monoecious Fagaceae Quercus suber. The results here depicted constitute a step towards the understanding of the molecular mechanisms involved in unisexual flower development in C. sativa, also suggesting that the ABCDE model for flower organ identity may be molecularly conserved in the predominantly monoecious Fagaceae family.

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Article
The Dynamics of Flower Development in Castanea sativa Mill.
Ana Teresa Alhinho 1, Miguel Jesus Nunes Ramos 2, Sofia Alves 2, Margarida Rocheta 2,
Leonor Morais-Cecílio 2, JoséGomes-Laranjo 3, Rómulo Sobral 1, * and Maria Manuela Ribeiro Costa 1, *


Citation: Alhinho, A.T.; Ramos,
M.J.N.; Alves, S.; Rocheta, M.;
Morais-Cecílio, L.; Gomes-Laranjo, J.;
Sobral, R.; Costa, M.M.R. The
Dynamics of Flower Development in
Castanea sativa Mill. Plants 2021,10,
1538. https://doi.org/10.3390/
plants10081538
Academic Editor: Cristina Ferrandiz
Received: 7 July 2021
Accepted: 23 July 2021
Published: 27 July 2021
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Copyright: © 2021 by the authors.
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conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1BioISI—Biosystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Centre,
University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; b7682@bio.uminho.pt
2LEAF—Linking Landscape, Environment, Agriculture and Food—Institute of Agronomy,
University of Lisbon, Tapada da Ajuda, 1359-017 Lisboa, Portugal; mramos@isa.ulisboa.pt (M.J.N.R.);
salves@isa.ulisboa.pt (S.A.); rocheta@isa.ulisboa.pt (M.R.); lmorais@isa.ulisboa.pt (L.M.-C.)
3CITAB—Centre for the Research and Technology of Agro-Environmental and Biological Sciences,
Universidade of Trás os Montes and Alto Douro, Quinta de Prados, 5000-801 Vila Real, Portugal;
jlaranjo@utad.pt
*Correspondence: romulosobral@bio.uminho.pt (R.S.); manuela.costa@bio.uminho.pt (M.M.R.C.)
Abstract:
The sweet chestnut tree (Castanea sativa Mill.) is one of the most significant Mediterranean
tree species, being an important natural resource for the wood and fruit industries. It is a monoecious
species, presenting unisexual male catkins and bisexual catkins, with the latter having distinct male
and female flowers. Despite the importance of the sweet chestnut tree, little is known regarding
the molecular mechanisms involved in the determination of sexual organ identity. Thus, the study
of how the different flowers of C. sativa develop is fundamental to understand the reproductive
success of this species and the impact of flower phenology on its productivity. In this study, a C. sativa
de novo transcriptome was assembled and the homologous genes to those of the ABCDE model for
floral organ identity were identified. Expression analysis showed that the C. sativa B- and C-class
genes are differentially expressed in the male flowers and female flowers. Yeast two-hybrid analysis
also suggested that changes in the canonical ABCDE protein–protein interactions may underlie the
mechanisms necessary to the development of separate male and female flowers, as reported for
the monoecious Fagaceae Quercus suber. The results here depicted constitute a step towards the
understanding of the molecular mechanisms involved in unisexual flower development in C. sativa,
also suggesting that the ABCDE model for flower organ identity may be molecularly conserved in
the predominantly monoecious Fagaceae family.
Keywords: Castanea sativa; Fagaceae; homeotic genes; ABCDE model; monoecy; unisexuality
1. Introduction
Development of unisexual male and female flowers has been regarded as a successful
mechanism to decrease inbreeding by promoting cross-pollination and gene fluidity [
1
].
The development of unisexual flowers has evolved, independently, multiple times, in some
families being the predominant reproductive strategy. Such is the case of the Fagaceae, a
large plant family comprising more than 900 species that constitute an important economic
resource and are pivotal to the dynamics of the forest ecosystems [2].
In the Fagaceae, the development of unisexual male and female flowers has been
studied in Quercus suber (cork oak) [
3
–
5
]. Sobral and Costa [
4
] have shown that changes
in the dynamics of the ABCDE model of flower organ identity may occur during the
development of the male and female flowers of Q. suber. The canonical ABCDE model
postulates that the development of the flower reproductive organs (stamens or carpels) is
controlled by distinct classes of genes: stamen identity is determined by the interaction
of B-, C-, and E-class transcription factors, whereas C-, D-, and E-class gene activity
specify carpel identity [
6
–
11
]. The ABCDE genes belong, predominantly, to the highly
conserved MADS-box family. These genes encode for transcription factors with four
Plants 2021,10, 1538. https://doi.org/10.3390/plants10081538 https://www.mdpi.com/journal/plants

Plants 2021,10, 1538 2 of 17
domains: MADS (M), intervening (I), keratin-like (K), and C-terminal (C) [
12
]. The MADS
domain, which is the DNA-binding domain, is the most conserved, conferring these
proteins the ability to bind to specific DNA motifs, the CArG-boxes [
12
,
13
]. The I and K
domains are involved in the mediation and establishment of multimeric protein–protein
complexes [
14
]. Although the C-terminal domain of the MADS-box proteins is much
less conserved, it contains lineage specific motifs that are functionally important. In the
specific case of B-class proteins, their stability and ability to bind CArG-boxes is dependent
on an obligatory heterodimerisation [
15
–
18
]. In cork oak, the expression of one of the
B-class genes, QsPISTILLATA (QsPI), determines the development of the male flower by
establishing a complex with other B-class and C-class proteins. In female flowers, complete
lack of QsPI expression disables the obligatory heterodimerisation of the B-class protein
complex, thus promoting carpel development [4].
The male and female unisexual flowers of the Fagaceae species are typically unisexual
by inception and develop at different periods during the growing season [
4
,
19
–
21
]. Castanea
and Lithocarpus species present two distinct inflorescences, unisexual male catkins and
bisexual catkins, the latter bearing functionally distinct male and female flowers [
22
,
23
]
suggesting different mechanisms controlling the development of unisexual flowers within
the Fagaceae. Castanea sativa Mill. is a deciduous Fagaceae species, distributed throughout
the Mediterranean region, spreading from the Caucasus to Portugal and from Southern
England to the southern part of the Iberian Peninsula [
24
]. It is monoecious, bearing two
types of inflorescences: unisexual male catkins that develop in the leaf axis of the current
season growth, and bisexual catkins that develop near the terminal end of the shoot. Each
unisexual male catkin is composed by staminate flowers gathered in glomerules of 3 to
7 flowers each, in an average number of 40 glomerules per catkin [
25
]. Each individual male
flower has four to six tepals and 8 to 12 stamens [
26
]. Female inflorescences appear singly
or in two or three clusters at the base of the bisexual catkins. Each inflorescence usually has
three flowers [
26
,
27
]. Each female flower typically presents six to eight styles, emerging
from enclosed scales. The ovaries have five to seven loci with two viable ovules each,
although usually only one of the ovules becomes a mature seed [
23
,
27
,
28
]. The stigmatic
region, which is exposed during the receptivity period, is covered with secretory cells,
which facilitates pollen adhesion and germination [
26
,
28
]. C. sativa presents a protandrous
habit, in which unisexual catkins mature before the bisexual ones, and pollen from the
unisexual catkin is released before the female flowers are fully receptive: this delay between
pollen shedding and female flower maturation takes more than two weeks [
26
,
29
]. Cross-
pollination is required to ensure fertilisation and fruit development due to gametophytic
self-incompatibility and morphological male sterility of some cultivars [27,30].
Most genetic resources related to the Castanea genus are derived from studies per-
formed in its close relative Castanea mollissima (Chinese chestnut), whose transcriptome
and whole-genome have been recently released [
31
,
32
]. However, there is still no available
transcriptomic data of C. sativa, despite its economic and ecological importance. Thus, the
molecular mechanisms controlling the development of the C. sativa unisexual and bisexual
catkins are still unclear, and it is not known to what extent these regulatory mechanisms
are comparable to what has been described for other Fagaceae species. In the present
study, we obtained a comprehensive transcriptome of C. sativa and studied the dynamics
of BCE-class gene expression and BCE-class protein complexes during the establishment of
the male and female flowers of C. sativa.
2. Results
2.1. Castanea sativa ‘Judia’ Cultivar Flowering Phenology
Castanea sativa is a monoecious species with an accentuated protandrous habit, as
unisexual male catkins and bisexual catkins develop with a significant delay. In this study,
a detailed phenological characterisation of the C. sativa ‘Judia’ cultivar was performed.
The ‘Judia’ cultivar was chosen because it is a predominant cultivar in Portugal due
to its high-quality fruits and productivity [
30
]. Unisexual male catkins emerge during

Plants 2021,10, 1538 3 of 17
spring in the axils of the new leaves that developed within the bursting axillary buds
(
Figure 1A,B
). At this stage of development, the glomerules are still covered by bracts.
As spring progresses, both the shoot and the unisexual male catkins elongate exposing
the glomerules (
Figure 1C,D
). At the same time, bisexual catkins start developing at the
terminal end of the new shoot, with no clear observable separation between female and
male flowers (
Figure 1E,F
). The development of the female and male organs in the bisexual
catkin is asynchronous as the male flower is the first to be identifiable. Towards the
beginning of summer, the glomerules in the unisexual male catkins are fully exposed as the
bracts start to recede, the anthers are visible, and pollen grains start to develop (
Figure 1G
,
white arrow). During the same period, the separation between the male and female flowers
becomes clearer in the bisexual catkin (Figure 1G, blue arrow, and detailed in
Figure 1H
(male flower); Figure 1G, pink arrow, and detailed in Figure 1I (female flower)). The styles
of the female flowers are not receptive (Figure 1J, pink arrow) when the pollen from the
unisexual male catkins is shed (Figure 1K). Thus, there is a temporal separation between
pollen shedding of the unisexual male catkin and the period in which female flower is
receptive (Figure 1L). The male flower in the bisexual catkin is not mature (Figure 1M)
during female flower receptivity. During mid-summer and after pollen shedding, the
unisexual male catkins start to acquire a yellow colour and fall. At this stage, pollen
shedding occurs in the male flower of the bisexual catkin, while the female flowers have
already been fertilised and start to swell (Figure 1N).
Plants 2021, 10, x FOR PEER REVIEW 3 of 18
a detailed phenological characterisation of the C. sativa ‘Judia’ cultivar was performed.
The ‘Judia’ cultivar was chosen because it is a predominant cultivar in Portugal due to its
high-quality fruits and productivity [30]. Unisexual male catkins emerge during spring in
the axils of the new leaves that developed within the bursting axillary buds (Figure 1A,B).
At this stage of development, the glomerules are still covered by bracts. As spring
progresses, both the shoot and the unisexual male catkins elongate exposing the
glomerules (Figure 1C,D). At the same time, bisexual catkins start developing at the
terminal end of the new shoot, with no clear observable separation between female and
male flowers (Figure 1E,F). The development of the female and male organs in the
bisexual catkin is asynchronous as the male flower is the first to be identifiable. Towards
the beginning of summer, the glomerules in the unisexual male catkins are fully exposed
as the bracts start to recede, the anthers are visible, and pollen grains start to develop
(Figure 1G, white arrow). During the same period, the separation between the male and
female flowers becomes clearer in the bisexual catkin (Figure 1G, blue arrow, and detailed
in Figure 1H (male flower); Figure 1G, pink arrow, and detailed in Figure 1I (female
flower)). The styles of the female flowers are not receptive (Figure 1J, pink arrow) when
the pollen from the unisexual male catkins is shed (Figure 1K). Thus, there is a temporal
separation between pollen shedding of the unisexual male catkin and the period in which
female flower is receptive (Figure 1L). The male flower in the bisexual catkin is not mature
(Figure 1M) during female flower receptivity. During mid-summer and after pollen
shedding, the unisexual male catkins start to acquire a yellow colour and fall. At this stage,
pollen shedding occurs in the male flower of the bisexual catkin, while the female flowers
have already been fertilised and start to swell (Figure 1N).
Figure 1. Flowering phenology of Castanea sativa. (A,B) In spring, following bud burst, the unisexual male catkins
emerge in the axils of new leaves (white arrows). (C,D) As spring progresses, bracts start to open and expose the
glomerules. (E,F) Bisexual catkin, with no clear distinction between the female and male flowers. (G) Shoot from June, in
which the unisexual catkins reach their final stage of development (white arrows) and the differentiation between male
(blue arrow, detailed in (H)) and female flowers (pink arrow, detailed in (I)) of the bisexual catkin is visible. (H) Detail of
the male flower of the bisexual catkin. (I) Detail of the female flower of the bisexual catkin. (J) Developed bisexual catkin,
with individualised male and female flowers (blue and pink arrows, respectively). (K) Glomerule of the unisexual male
catkin, with anthers exposed. (L) Receptive female flower with exposed stigma. (M) Developed glomerule of the male
flower of the bisexual catkin. (N) Shoot from July, in which the unisexual catkin is already turning yellow and dehiscent,
having shed its pollen, and the female flower is swelling in order to develop the bur.
2.2. Castanea sativa De Novo Transcriptome Analysis
Despite the C. sativa economic and ecological importance, it is still not clear which
molecular mechanisms are employed to control the flower phenology in this species as no
genomic/transcriptomic resources were available. Therefore, the assembly of a de novo C.
Figure 1. Flowering phenology of Castanea sativa.
(
A
,
B
) In spring, following bud burst, the unisexual male catkins
emerge in the axils of new leaves (white arrows). (
C
,
D
) As spring progresses, bracts start to open and expose the glomerules.
(
E
,
F
) Bisexual catkin, with no clear distinction between the female and male flowers. (
G
) Shoot from June, in which the
unisexual catkins reach their final stage of development (white arrows) and the differentiation between male (blue arrow,
detailed in (
H
)) and female flowers (pink arrow, detailed in (
I
)) of the bisexual catkin is visible. (
H
) Detail of the male
flower of the bisexual catkin. (
I
) Detail of the female flower of the bisexual catkin. (
J
) Developed bisexual catkin, with
individualised male and female flowers (blue and pink arrows, respectively). (
K
) Glomerule of the unisexual male catkin,
with anthers exposed. (
L
) Receptive female flower with exposed stigma. (
M
) Developed glomerule of the male flower of the
bisexual catkin. (
N
) Shoot from July, in which the unisexual catkin is already turning yellow and dehiscent, having shed its
pollen, and the female flower is swelling in order to develop the bur.
2.2. Castanea sativa De Novo Transcriptome Analysis
Despite the C. sativa economic and ecological importance, it is still not clear which
molecular mechanisms are employed to control the flower phenology in this species as no
genomic/transcriptomic resources were available. Therefore, the assembly of a de novo C.
sativa transcriptome was vital to lay the foundations for molecular studies in this species.
To obtain the transcriptome of C. sativa, we isolated total RNA from male flowers from the
unisexual catkin and male and female flowers from the bisexual catkin, leaves, and buds at

Plants 2021,10, 1538 4 of 17
different developmental stages during the plant life cycle. The RNA samples were pooled
together and sequenced using Illumina technology to generate 2
×
150 bp paired-end reads.
After trimming, 134,286,842 sequences were obtained and used for de novo transcriptome
assembly. A pipeline for de novo transcriptome assembly and processing proposed by
Chabikwa et al. [
33
] was implemented, resulting in 164,926 contigs (Table S1). To evaluate
the reliability and quality of this assembly, we mapped the reads used to generate the de
novo transcriptome back to the transcriptome using bowtie2 [
34
]. The alignment rate of
the input RNA-Seq reads was 97.49%, whereas alignment with the C. mollissima genome
yielded an overall alignment rate of 62.93%.
After redundancy removal, 32,871 transcripts were obtained, with an average length
of 906 bp, an N50 value of 1230 bp, and a GC percentage of 44% (Table S1). BUSCO is a
tool that quantitatively assesses the quality and coverage of a transcriptome on the basis of
evolutionarily informed expectations of gene content from eukaryotic genomes [
35
]. In the
C. sativa transcriptome, 94.1% of the BUSCOS had a complete representation, indication
a high-quality assembly, with 86.67% single copy genes (Table S2). The 32,871 transcript
sequences were uploaded to Blast2GO [
36
], and blastx was performed against the NCBI nr
database with an E value cut off of 1e-3; 29,607 sequences had blast hits, and 3264 had no
hits. GO annotation categorised the transcripts in different functional groups according to
cellular components, biological process, and molecular function (Figure S1).
2.3. ABCDE MADS-Box Transcription Factors Were Conserved in Castanea sativa
In cork oak, a closely related Fagaceae, the identity of the female and male sexual
organs is dependent on the spatial activity of the ABCDE MADS-box genes [
4
]. It is not
clear, however, as to the way in which in other Fagaceae, such as C. sativa, the ABCDE
model is involved in the determination of flower unisexuality.
To check if sexual organ identity development in sweet chestnut is controlled by
MADS-box genes, we screened the de novo transcriptome for homologs of Arabidopsis
thaliana or Solanum lycopersicum MADS-box proteins (SlTM6 was used as a query be-
cause there is no TM6 homolog in A. thaliana). The blast queries included homologs for
genes belonging to A-class (AtAPETALA1), B-class (AtPI, SlTM6, and AtAPETALA3), C-
and D-classes (AtAGAMOUS,AtSHATTERPROOF1-2, and AtSEEDSTICK), and E-class
(AtSEPALATTA1-4). The phylogeny of the C. sativa ABCDE MADS-box proteins was in-
ferred using homologs of other angiosperms, and, whenever possible, homologs from an
ancestral angiosperm (Amborella trichopoda) and a gymnosperm (Pinus radiata). On the
basis of multiple alignments, we were able to conclude that all the retrieved sequences
were complete and displayed the four conserved domains (M-, I-, K-, and C-terminals)
(Figure S2).
In the A-class (Figure 2A), CsaAPETALA1 (CsaAP1) clusters with AP1 proteins from
other perennials, in the same clade that includes AP1-like proteins from C. mollissima,
Q. suber, Betula pendula, and Corylus avellana, species belonging to the order Fagales. The
B-class phylogroup (Figure 2B) can be divided into three lineages: PI, euAP3, and pale-
oAP3 [
37
]. The PI and paleoAP3 lineages arose from a duplication event that took place
before the emergence of angiosperms, whereas a later duplication event that took place at
the base of higher dicots originated the euAP3 lineage [
37
] (reviewed in [
38
]). One sweet
chestnut homolog was identified in each lineage (CsaPI, CsaAP3, and CsaTM6) clustering
with homologs of C. mollissima, Quercus robur, Q. suber, and Quercus rubra, all of them
monoecious Fagaceae. The PI lineage is characterised by a PI motif that is essential for
protein function [
37
]. A comparative analysis of the PI motif between AtPI and CsaPI
revealed that CsaPI has a complete PI motif in its C-terminal domain that seems to be
conserved in the Fagaceae (Figure S3A). The core difference between the paleoAP3 and
euAP3 lineages lies in the motifs contained in the C-terminal domain (Figure S2B) [
37
].
Comparison of the euAP3 motif of A. thaliana and C. sativa homologs suggests a partial
conservation of the euAP3 motif in CsaAP3, similar to what happens in other Fagaceae
(Figure S3). Similarly, there is a partial conservation of the paleoAP3 motif in CsaTM6.

Plants 2021,10, 1538 5 of 17
This motif is totally conserved in C. sativa and C. mollissima, but there is one amino acid
difference between proteins of the Castanea and Quercus genus (QsTM6) (Figure S3C).
AB
AP3 clade
TM6 clade
PI clade
CD
AG clade
SHP clade
STK clade
SEP1/2 clade
SEP4 clade
SEP3 clade
Figure 2. Phylogenetic profiling of the Castanea sativa ABCDE genes.
(
A
) A-class lineage; (
B
) B-class lineage; (
C
) C- and
D-class lineages; (
D
) E-class lineage. Phylogenies were inferred using the maximum likelihood method (1000 replicates).
The percentage of replicate trees in which the associated taxa clustered together is shown next to the branch nodes. The
JTT method was used to compute evolutionary distances (number of aminoacids per site). The accession numbers for
the proteins used in this analysis are presented in Table S3. Fagaceae species are highlighted in the blue box. The protein
sequences used in this analysis were retrieved from the following species: Castanea mollissima (Cm), Quercus suber (Qs),
Quercus robur (Qr), Quercus rubra (Qru), Fagus crenata (Fc), Betula pendula (Bp), Corylus avellana (Ca), Juglans regia (Jr), Malus
domestica (Md), Prunus persica (Pp), Actinidia chinensis (Ac), Populus trichocarpa (Pt), Vitis vinifera (Vv), Arabidopsis thaliana
(At), Citrus unshiu (Cu), Solanum lycopersicum (Sl), Cucumis sativus (Cs), Anthirrinum majus (Am), Medicago truncatula (Mt),
Glycine max (Gm), Oryza sativa (Os), Zea mays (Zm), Pinus radiata (Pr), Amborella trichopoda (Atr).
The AG subfamily also underwent several duplication events throughout evolution:
one duplication event originated the C and D lineages before the radiation of extant
angiosperms [
13
]. The phylogenetic analysis for the C- and D-class proteins (
Figure 2C
)

Plants 2021,10, 1538 6 of 17
placed CsaAG in the same clade as C. mollissima and Q. suber AGAMOUS homologs.
CsaSHP was also placed in the same clade as the homologs of the closely related Fagaceae
Q. suber and Q. rubra (Figure 2C). There are two characteristic motifs (I and II) in the C-
terminal domain of AG proteins [
39
]. CsaAG seems to have a partial conservation of these
motifs, compared to its A. thaliana counterpart, and a complete amino acid conservation
within the Fagaceae (Figure S2D). There is also partial conservation between CsaSHP
and AtSHP AG motifs I and II, and a full conservation between the Fagaceae in motif I
(
Figure S2E
). However, the same pattern is not followed regarding the AG motif II, where
there is a complete deletion in QsSHP, which might suggest functional divergence.
The E-class comprises the SEPALLATA genes, which act as co-factors in the protein
complexes that specify the identity of the different floral organs [
40
]. In the C. sativa
transcriptome, four SEP homologs were identified (Figure 2D). Regarding the SEP1/2
lineage, both the CsaSEP1 and CsaSEP2 are closely related to the proteins of C. mollissima
and Q. suber. CsaSEP3 also clusters with other Fagales; however, it is related to a closer
degree with AtSEP3. CsaSEP4 is located in the same clade as SEP4 homologs of C. mollissima,
Q. suber, and Q. robur.
2.4. C. sativa BCE-Like Gene Expression in Unisexual and Bisexual Catkins
During floral developmental, the coordinated spatial and temporal regulation of the
MADS-box gene expression is pivotal for proper floral organ development (reviewed
in [
40
]). The canonical ABCDE model postulates that stamen identity is determined by
the expression of B-, C-, and E-class genes in the third whorl, whereas carpel identity is
determined by expression of C- and E-class genes in the centre floral whorl (reviewed
in [13]).
An expression analysis, by qRT-PCR, of the sweet chestnut BCE-like gene homologs
was conducted using samples that included male unisexual catkins, as well as male and
female flowers from the bisexual catkins, at different stages of development. The develop-
mental stages were collected after the emergence of the unisexual male catkin primordia
until their full development (MF S1–S4, represented in Figure 1A–D, respectively); after the
emergence of bisexual catkins (BP, Figure 1E–F), in which there is no observable separation
between the male and female flowers, and after complete separation and developed male
(MB 1, represented in Figure 1H, and MB 2, represented in Figure 1M) and female flowers
(FB 1, represented in Figure 1I, and FB 2, represented in Figure 1L).
The B-class genes, CsaAP3,CsaPI and CsaTM6 (Figure 3A–C), present similar expres-
sion patterns during the unisexual male flower development, increasing steadily from
stages MF-S1 to MF-S4 (Figure 3A). The expression of CsaAP3,CsaPI and CsaTM6 is low
during bisexual catkin primordia development (BP) and in early stages of its male and
female flowers development (MB-S1 and FB-S1), but the expression peaks in the male
portion of the bisexual catkin in stage MB-S2 (Figure 3A–C). There is expression of CsaPI
in the female structure of the bisexual catkin in stage FB-S2, although it is not detected
in stage FB-S1 (Figure 3B). The expression of the three B-class genes in female flowers
led to a more thorough morphologic and histological analysis of these flowers, which
revealed the presence of stamen-like structures fused at the base of the styles, concealed
by the perianth (Figure 4A–C). Histological analysis of the male flowers revealed that no
carpelloid structures can be found in these flowers (Figure 4D).

Plants 2021,10, 1538 7 of 17
Plants 2021, 10, x FOR PEER REVIEW 7 of 18
Figure 3. Expression analysis of C. sativa BCE homolog genes in Castanea sativa flowers. (A) CsaAP3; (B) CsaPI; (C)
CsaTM6; (D) CsaAG; (E) CsaSHP; (F) CsaSEP2; (G) CsaSEP3; (H) CsaSEP4. Error bars indicate standard deviation (SD) of
three biological and technical replicates. CsaPP2AA3 was used as a reference gene. MF S1—unisexual male catkin primor-
dia appearing in the axils of leaves of bursting buds (Figure 1A); MF S2—immature unisexual male catkins, starting to
arise from bursting buds (Figure 1B); MF S3—mature unisexual male catkins (Figure 1C); MF S4—unisexual male catkin
with developed glomerules, at a later stage of development (Figure 1D); BP—bisexual catkin primordia, with no definition
between male and female flowers (Figure 1E,F); MB S1—male flowers of the bisexual catkin, early stage (Figure 1H); FB
S1—female flower of the bisexual catkin, early stage (Figure 1I); MB S2—male flower of the bisexual catkin, fully devel-
oped (Figure 1M); FB S2—female flower of the bisexual catkin, fully developed (Figure 1L).
Figure 3. Expression analysis of C. sativa BCE homolog genes in Castanea sativa flowers
. (
A
)CsaAP3; (
B
)CsaPI; (
C
)
CsaTM6; (
D
)CsaAG; (
E
)CsaSHP; (
F
)CsaSEP2; (
G
)CsaSEP3; (
H
)CsaSEP4. Error bars indicate standard deviation (SD) of
three biological and technical replicates. CsaPP2AA3 was used as a reference gene. MF S1—unisexual male catkin primordia
appearing in the axils of leaves of bursting buds (Figure 1A); MF S2—immature unisexual male catkins, starting to arise
from bursting buds (Figure 1B); MF S3—mature unisexual male catkins (Figure 1C); MF S4—unisexual male catkin with
developed glomerules, at a later stage of development (Figure 1D); BP—bisexual catkin primordia, with no definition
between male and female flowers (Figure 1E,F); MB S1—male flowers of the bisexual catkin, early stage (Figure 1H); FB
S1—female flower of the bisexual catkin, early stage (Figure 1I); MB S2—male flower of the bisexual catkin, fully developed
(Figure 1M); FB S2—female flower of the bisexual catkin, fully developed (Figure 1L).

Plants 2021,10, 1538 8 of 17
Plants 2021, 10, x FOR PEER REVIEW 8 of 18
Figure 4. Female flowers from the ‘Judia’ cultivar present stamen-like structures. (A,B) Detail of the stamen-like structures
at the base of the styles of female flowers—the perianth was removed to expose these organs (red arrows). (C) Longitudinal
section of a female flower, where it is possible to see the stamen-like organ fused with the style (red arrow). (D) Transversal
section of a male flower, with no evidence of stylar-like organs.
CsaSHP expression follows a similar pattern to that of B-class genes, presenting low
expression in early stages of male flower development and peaking at MF-S4 (Figure 3E).
CsaSHP expression is low in bisexual primordia and in early stages after the separation of
male and female structures, but increases in stages S2 for both tissues (MB-S2 and FB-S2),
being higher in the male flowers than in the female flowers. The fact that CsaSHP is ex-
pressed also in the male flowers suggests that this gene might have retained a C-function
after the duplication event that originated the C and D lineages. CsaAG is significantly
more expressed in unisexual male flowers, mainly in the later stage (MF-S4), and in the
bisexual catkins its expression follows a pattern similar to that of CsaSHP, with low ex-
pression levels in the early developmental stages, and higher in later stages of develop-
ment (Figure 3D). CsaSTK expression was not detected in the flowers at the chosen devel-
opmental stages.
E-class genes CsaSEP2 (Figure 3F) and CsaSEP4 (Figure 3H) present low expression
levels in early stages of unisexual male flower development, peaking in MF-S4. CsaSEP3
(Figure 3G) shows an inverse expression pattern, being significantly higher during early
development (MF S1) and decreasing in later stages. In the bisexual catkin primordia (BP)
all the SEP-like genes are expressed, and upon early male and female flower development,
their expression is higher in female flowers (FB S1) than in male flowers (MB S1). CsaSEP1
was not detected in any of the flower tissues.
Overall, CsaAG, CsaSHP, CsaAP3, CsaPI, CsaTM6 and CsaSEPs showed higher expres-
sion in the unisexual male catkin flowers and the male flower of the bisexual catkin than
in female flowers, particularly at later stages of development.
Figure 4.
Female flowers from the ‘Judia’ cultivar present stamen-like structures. (
A
,
B
) Detail of
the stamen-like structures at the base of the styles of female flowers—the perianth was removed to
expose these organs (red arrows). (
C
) Longitudinal section of a female flower, where it is possible to
see the stamen-like organ fused with the style (red arrow). (
D
) Transversal section of a male flower,
with no evidence of stylar-like organs.
CsaSHP expression follows a similar pattern to that of B-class genes, presenting low
expression in early stages of male flower development and peaking at MF-S4 (Figure 3E).
CsaSHP expression is low in bisexual primordia and in early stages after the separation
of male and female structures, but increases in stages S2 for both tissues (MB-S2 and
FB-S2), being higher in the male flowers than in the female flowers. The fact that CsaSHP is
expressed also in the male flowers suggests that this gene might have retained a C-function
after the duplication event that originated the C and D lineages. CsaAG is significantly more
expressed in unisexual male flowers, mainly in the later stage (MF-S4), and in the bisexual
catkins its expression follows a pattern similar to that of CsaSHP, with low expression levels
in the early developmental stages, and higher in later stages of development (Figure 3D).
CsaSTK expression was not detected in the flowers at the chosen developmental stages.
E-class genes CsaSEP2 (Figure 3F) and CsaSEP4 (Figure 3H) present low expression
levels in early stages of unisexual male flower development, peaking in MF-S4. CsaSEP3
(Figure 3G) shows an inverse expression pattern, being significantly higher during early
development (MF S1) and decreasing in later stages. In the bisexual catkin primordia (BP)
all the SEP-like genes are expressed, and upon early male and female flower development,
their expression is higher in female flowers (FB S1) than in male flowers (MB S1). CsaSEP1
was not detected in any of the flower tissues.
Overall, CsaAG,CsaSHP,CsaAP3,CsaPI,CsaTM6 and CsaSEPs showed higher expres-
sion in the unisexual male catkin flowers and the male flower of the bisexual catkin than in
female flowers, particularly at later stages of development.
2.5. Dimerisation of C. sativa BCE-Like Proteins
According to the canonical ABCDE model, stamen development depends on the
interaction of B-, C-, and E-class proteins, whereas carpel development depends on the
interaction of C- and E-class proteins [
41
,
42
]. Different dynamics of the ABCDE model

Plants 2021,10, 1538 9 of 17
in the Fagaceae Q. suber, namely, the interaction of B-class QsAP3 and QsPI with C-class
QsSHP, and not QsAG (C-class), may be one of the factors underlying unisexual flower
development in this species [
4
]. It is not clear if redeployment of the canonical ABCDE
model interactions could be the mechanism behind the development of all the unisexual
flowers in C. sativa.
Dimerisation of C. sativa BCE-like proteins was assessed in a yeast-two-hybrid experi-
ment, in which the coding regions of the C. sativa B-, C-, and E-like genes were fused to
the activation or binding domain of the GAL4 transcription factor. Interactions between
C. sativa B-, C-, and E-like proteins are compiled in Figure 5.
Figure 5. Interaction between Castanea sativa BCE-like proteins.
Double yeast transformations of C. sativa BCE-like
proteins fused to GAD4-activation domain (AD) and GAL4-binding domain (BD) were performed and tested for plasmid
presence by growing cells in synthetic defined (SD) medium without tryptophan and lecucine (-W-L). Interacting capacity
was tested in SD medium without aminoacids supplemented with 3-aminotriazole (tryptophan, leucine, histidine). Inter-
action strength was evaluated through sequential dilutions (1:10, 1:100, 1:1000, 1:10,000, 1:100,000). Each cropped image
corresponds to a matching single plate in which the double transformants were selected.
An important trademark of the ABCDE model is the functional obligatory heterodimeri-
sation between the B-class proteins. In C. sativa, the obligatory heterodimerisation of AP3
and PI-like proteins is likely to be conserved, as CsaPI and CsaAP3 proteins were able
to dimerise and activate the reporter gene HISTIDINE3. CsaAP3 also interacted with its
closest relative CsaTM6, but there was no interaction detected between CsaPI and CsaTM6.
Interaction between B- and E-class proteins seems to be conserved in C. sativa, as all B-class
proteins were able to interact with CsaSEP2, CsaSEP3, or CsaSEP4. CsaSEP1 protein was

Plants 2021,10, 1538 10 of 17
not used in this assay because CsaSEP1 expression was not detected in flower tissues, as
mentioned previously. The B-class proteins CsaPI and CsaAP3 were also able to interact
with the C-class protein CsaSHP, but not with CsaAG (Figure 5).
The other C-class protein, CsaAG, was only able to interact with the E-class proteins
CsaSEP2 and CsaSEP3, but the activation of the gene reporter was not sufficient to allow
yeast growth in higher order dilutions, suggesting a weaker interaction between these
proteins. The expression of CsSHP in male and female flowers combined with the non-
canonical dimerisation of CsaSHP with B-class proteins suggests that CsaSHP might have
retained a C-class function in the determination of floral reproductive organs.
3. Discussion
Changes to the ABCDE model dynamics is proposed as one of the factors controlling
the development of unisexual flowers by inception in several families, including the
predominantly monoecious Fagaceae [
4
,
33
–
35
]. In cork oak, several ABCDE gene homologs
are differentially expressed in male and female flowers, suggesting that floral organ identity
in this species is controlled by rearrangements in the expression dynamics of these genes.
In the monoecious C. sativa, a similar scenario could also explain the development of its
unisexual flowers. Therefore, it is of particular importance to characterise B- and C-function
genes that specify male and female organ identity, in order to clarify the mechanism of
sexual organ determination in C. sativa. In the present work, several genes homologous
to the ABCDE model genes were identified and their role in the establishment of C. sativa
unisexual flowers was assessed.
Ade novo transcriptome of C. sativa was assembled and surveyed for ABCDE model
genes homologs. According to this model, female flower identity is established by the
coordinated activity of C- and E-class genes, whereas male flower development depends of
B-, C-, and E-class genes [
13
]. In the present study, three B-class genes were identified and
classified according to their phylogenetic clustering (CsaAP3,CsaPI and CsaTM6). B-class
genes are expressed in the second and third whorl of the flower meristem, being involved
in petal and stamen identity, respectively [
13
]. In C. sativa, the temporal expression profile
for all B-class genes is similar, being higher in male flowers from the unisexual catkin and
in the male flower of the bisexual catkin, and significantly lower in the female flower.
The expression pattern presented by the B-class genes CsaTM6 and CsaAP3 in C. sativa
is similar to its close relative Q. suber:QsTM6.1/2 and QsAP3 present high expression levels
in male flowers, being expressed in female flowers to a lesser extent [
4
]. However, this
pattern is not mimicked by CsaPI, as there is expression of this gene in female flowers, in
late developmental stages, which does not occur in Q. suber. Reports of hermaphroditism in
C. sativa female flowers [
26
,
36
–
40
] suggest that the rudimentary stamens are sterile [
27
,
36
].
A pollen germination assay might be required to assess the fertility of these rudimentary
stamens in the presently studied ‘Judia’ cultivar. The presence of these stamen-like organs
in female flowers is not exclusive of C. sativa, having been also detected in other species of
the Castanea genus. Morphologic observations in C. mollissima revealed the presence of 12
underdeveloped staminodes with short filaments and visible anthers capable of producing
pollen, which did not dehisce [
41
,
42
]. In the Chinese chinquapin (Castanea henryi), female
flowers experience a period of hermaphroditism, as 12 stamen primordia also develop
during carpel development, arresting their elongation as the stigma enlarges [
43
]. It is
thus likely that female flowers in the Castanea genus are hermaphroditic at inception and
become unisexual by abortion or arrested growth of male organs. This is contrary to what
was reported for other Fagaceae, in which the flowers are unisexual by inception as no
aborted organs of the opposite sex are detected [
4
,
19
–
21
]. The expression of CsaPI and the
other B- and C-class genes in the female flowers is likely associated to the development
of the stamen-like structures in the female flowers of C. sativa. In the present study, CsaPI
was found to be able to interact with CsaAP3 and CsaTM6, which is indicative that the
obligatory heterodimerisation of B-class proteins is conserved also in this species. In the
other Fagaceae Q. suber, this interaction was also shown [
4
]. Homodimerisation of AP3

Plants 2021,10, 1538 11 of 17
homologs is also conserved in these two Fagaceae. However, the interaction between
QsAP3 and QsTM6 was not reported for Q. suber, while in C. sativa, CsaAP3 and CsaTM6
were able to interact. Thus, it is likely that different interactions between Fagaceae B-
class proteins might be species-specific. In A. thaliana, E-class genes have ubiquitous and
redundant functions in flower meristem identity and flower organ development in the four
whorls, as triple and quadruple mutants for these genes have floral organs converted into
sepals and leaf-like organs [
10
,
44
]. In C. sativa, E-class proteins might have their function
in flower organ identity conserved, as they are able to interact with B- and C-class proteins.
Thus, similarly to what was determined in the close relative Q. suber, the heterodimerisation
of B-class proteins and their interaction with C- and E-class proteins might be needed for
male flower development in C. sativa (Figure 6).
Plants 2021, 10, x FOR PEER REVIEW 12 of 18
and cucumber [4,48,49], indicative that the interactions between these proteins are rela-
tively conserved among species. On the other hand, C. sativa B-class proteins CsaAP3 and
CsaPI, but not CsaTM6, were also able to heterodimerise with C-class CsaSHP, as was
previously reported for Q. suber, suggesting a conservation of this non-canonical interac-
tion in the Fagaceae. The similar temporal expression patterns of CsaAG and CsaSHP, cou-
pled with the fact that there was no observed interaction between B-class proteins and
CsaAG, might indicate that CsaSHP may be required to act simultaneously with CsaAG in
order to complete the C-function, as was observed in other species [4,50].
The temporal expression of the ABCDE model homologous genes in C. sativa and the
ABCDE model interactome suggests that it is likely that the homeotic genes are involved
in the determination of the male flower identity, both in the unisexual and bisexual cat-
kins. However, the determination of unisexuality in the female flowers requires a different
explanation. It is possible that the spatial expression of CsaPI in the female flower could
be restricted to the non-reproductive and stamen whorls, allowing the development of
stamens in the third whorl and the carpel in the centre whorl (Figure 6). Analysis of the
spatial expression of CsaPI during the bisexual catkin primordia formation might help
unveil the role of this gene during female flower development. Furthermore, the mecha-
nisms that could be promoting the probable stamen abortion in this cultivar are still illu-
sive. Thus, RNA-Seq studies concerning C. sativa inflorescences will be pivotal to clarify
this hypothesis, as well as to identify genes exclusively expressed in female inflorescences
that might be involved in the genetic basis of their development. It is possible that some
of these genes are related to the developmental arrest of the stamens, as was reported in
transcriptomic studies in other species in which sex determination is a late event [51–54].
Figure 6. Representative model of the potential role of MADS-box transcription factors during the development of Castanea
sativa unisexual flowers. (A) Combinatorial action of B-class CsaPI/TM6/AP3 with C-class CsaSHP and E-class CsaSEPs
confers male identity to the unisexual male flowers. (B) Female identity is conferred by a combination of CsaSHP and
CsaSEPs. Unknown mechanisms, downstream of the MADS-box genes, may contribute to the development of staminodes
in female flowers (blue box, red arrows).
Clarification of the mechanisms that lead to flower unisexuality are of particular in-
terest in species of agronomical and ecological relevance, as it has biological implications,
affecting fructification, seed production, and species perpetuation. C. sativa constitutes a
particular case of variation of monoecy within the Fagaceae family, presenting unisexual
male catkins and bisexual catkins. Furthermore, unlike most Fagaceae, C. sativa female
Figure 6.
Representative model of the potential role of MADS-box transcription factors during the development of Castanea
sativa unisexual flowers. (
A
) Combinatorial action of B-class CsaPI/TM6/AP3 with C-class CsaSHP and E-class CsaSEPs
confers male identity to the unisexual male flowers. (
B
) Female identity is conferred by a combination of CsaSHP and
CsaSEPs. Unknown mechanisms, downstream of the MADS-box genes, may contribute to the development of staminodes
in female flowers (blue box, red arrows).
In the C. sativa transcriptome, one AG protein (CsaAG) was inferred, which clustered
in the euAG clade from the Fagaceae. In the PLENA lineage, one SHP-like protein was
identified (CsaSHP). Expression analysis of these genes in C. sativa flowers revealed that
CsaAG is mainly expressed in later developmental stages of male flowers, a pattern that has
also been observed in other species with unisexual flowers, such as Populus trichocarpa [
45
],
Q. suber [
4
], and B. pendula [
46
] with a strong association with pollen development. Unlike
CsaAG,CsaSHP is expressed during unisexual male flower development, in the bisexual
catkin primordia, and in the female and male flowers of the bisexual catkin. Similarly to
CsaSHP,QsSHP was also detected in both the male and female flowers [
4
], suggesting that
the Fagaceae SHP homologs, and not the AG homologs, could be acting as functional C-like
genes.
Regarding the C-class proteins interactions, CsaAG only dimerises with CsaSEP3
and CsaSEP2. Similar results were found in Q. suber [
4
] and Prunus mume [
47
], in which
AG-like proteins only interacted with SEP-like proteins. CsaSHP displayed interaction
with all SEP-like proteins, which was also observed in species such as Arabidopsis, cork
oak, and cucumber [
4
,
48
,
49
], indicative that the interactions between these proteins are
relatively conserved among species. On the other hand, C. sativa B-class proteins CsaAP3
and CsaPI, but not CsaTM6, were also able to heterodimerise with C-class CsaSHP, as was

Plants 2021,10, 1538 12 of 17
previously reported for Q. suber, suggesting a conservation of this non-canonical interaction
in the Fagaceae. The similar temporal expression patterns of CsaAG and CsaSHP, coupled
with the fact that there was no observed interaction between B-class proteins and CsaAG,
might indicate that CsaSHP may be required to act simultaneously with CsaAG in order to
complete the C-function, as was observed in other species [4,50].
The temporal expression of the ABCDE model homologous genes in C. sativa and the
ABCDE model interactome suggests that it is likely that the homeotic genes are involved in
the determination of the male flower identity, both in the unisexual and bisexual catkins.
However, the determination of unisexuality in the female flowers requires a different
explanation. It is possible that the spatial expression of CsaPI in the female flower could
be restricted to the non-reproductive and stamen whorls, allowing the development of
stamens in the third whorl and the carpel in the centre whorl (Figure 6). Analysis of
the spatial expression of CsaPI during the bisexual catkin primordia formation might
help unveil the role of this gene during female flower development. Furthermore, the
mechanisms that could be promoting the probable stamen abortion in this cultivar are still
illusive. Thus, RNA-Seq studies concerning C. sativa inflorescences will be pivotal to clarify
this hypothesis, as well as to identify genes exclusively expressed in female inflorescences
that might be involved in the genetic basis of their development. It is possible that some
of these genes are related to the developmental arrest of the stamens, as was reported in
transcriptomic studies in other species in which sex determination is a late event [51–54].
Clarification of the mechanisms that lead to flower unisexuality are of particular
interest in species of agronomical and ecological relevance, as it has biological implications,
affecting fructification, seed production, and species perpetuation. C. sativa constitutes a
particular case of variation of monoecy within the Fagaceae family, presenting unisexual
male catkins and bisexual catkins. Furthermore, unlike most Fagaceae, C. sativa female
flowers are not unisexual by inception. The likely temporary hermaphroditism in this
species could be considered a relaxation that is counterbalanced by an additional layer to
prevent inbreeding, which is the C. sativa gametophytic self-incompatibility. Thus, it is of
utmost importance to study the genetic mechanisms for unisexual flower development in
this species, which may differ significantly within the Fagaceae. The results here presented
show that, even though phylogenetically close, ABCDE model genes may have different
dynamics within distinct Fagaceae species.
4. Materials and Methods
4.1. De Novo Transcriptome Assembly
4.1.1. Plant Material
Tissue samples (leaves, buds, and flowers in several developmental stages) from
Castanea sativa M. were harvested at the germplasm bank located at UTAD (Vila Real
district, Portugal, 41.2863826,
−
7.7448634) from three adult trees from the ‘Judia’ cultivar
located in Lagoa (Vila Pouca de Aguiar district, Portugal, 41.6292217,
−
7.2960124). The
flower developmental stages were selected on the basis of the phenological stages described
in [
55
]. The collected tissues were frozen in liquid nitrogen and kept at
−
80
◦
C until RNA
extraction.
4.1.2. RNA Extraction
For de novo transcriptome assembly, total RNA was extracted from frozen tissue
samples of young and mature leaves, dormant and active buds, and male and bisexual
inflorescences in different stages of development. Samples were ground to powder using
liquid nitrogen, and the RNA was extracted using the CTAB/LiCl method [
56
], with
some modifications [
57
,
58
]. DNAse treatment was conducted using TURBO
™
DNase
(ThermoFischer, Waltham, MA, USA), following the manufacturer’s instructions, and
integrity was analysed using the Experion
™
RNA StdSens Analysis Kit (BioRad, Hercules,
CA, USA), prior to library preparation.

Plants 2021,10, 1538 13 of 17
4.1.3. Library Preparation and Sequencing
The individual RNA samples were pooled, and the cDNA libraries were prepared
using Illumina TruSeq stranded mRNA kit and sequenced at Fasteris S.A. (Plain-les-Ouates,
Switzerland), using Illumina technology to generate 2 ×150 bp paired-end reads.
4.1.4. De Novo Assembly of Castanea sativa Transcriptome
FastQC v0.11.8 [
59
] was applied to check the quality of the sequencing data. Low-
quality reads were filtered and Illumina adapters removed using bbduk from the package
bbtools [
60
]. All sequences with a quality score below 20 were removed and a forced
trimming of the first 15 bp was performed. Overrepresented polyG sequences were
removed using the option “literal”. Furthermore, in order to eliminate small reads that
were interfering with the sequence length distribution, we filtered all reads with a length
below 30 bp.
De novo transcriptome assembly was performed using Trinity (v. 2.9.1) [
61
,
62
] in a
Galaxy server (https://usegalaxy.org/, accessed on 26 July 2021), with default settings.
Assembly metrics were investigated using TransRate (v. 1.0.3) [
63
], a software that re-
ports basic statistics such as assembly score, number of contigs, and N50. To examine the
RNA-Seq read representation of the assembly, we mapped the reads used to generate the
de novo
assembly against the assembly using bowtie2 (v. 2.3.4.3) [
64
]. TransDecoder (Galaxy
Version 3.0.1), with default settings, was used to predict coding regions within the transcrip-
tome and remove redundancy. The single best open reading frame (ORF) per transcript
longer than 100 peptides was used. CD-HIT-EST (v. 4.7) [
65
] was used with 95% similarity
to reduce transcript redundancy and produce unique genes (henceforth “transcripts”).
Benchmarking Universal Single-Copy Orthologs (BUSCO) [
66
], part of OmicsBox (v. 1.2.3,
https://www.biobam.com/omicsbox, accessed on 26 July 2021), was used to validate the
transcriptome assembly. Functional annotation of the whole de novo transcriptome was
performed using Blast2GO (v. 5.2.5) [
67
]. The sequences were blasted against NCBI NR
protein database (March, 2020) and were scanned with InterPro and mapped against the
PSD, UniProt, Swiss-Prot, TrEMBL, RefSeq, GenPept, and PDB databases. The reads that
originated the transcriptome were deposited in NCBI Sequence Read Archive under the
accession number PRJNA744401.
4.2. Phylogenetic Analysis
Castanea sativa MADS-box transcript sequences were obtained using the blast+ soft-
ware [
68
] by performing a BLAST in the C. sativa de novo transcriptome using A. thaliana
sequences as query. Homologous protein sequences from other selected species were
obtained by performing a BLASTx at the NCBI database (https://www.ncbi.nlm.nih.gov/,
accessed on 26 July 2021), Hardwood Genomics (https://www.hardwoodgenomics.org/,
accessed on 26 July 2021) and URGI (https://urgi.versailles.inra.fr/, accessed on 26 July
2021). Protein sequences were aligned using Clustal Omega [
69
], and distances were
estimated using the Jones–Taylor–Thornton (JTT) model of evolution for a maximum likeli-
hood tree with the MEGA X software [
70
]. A phylogenetic tree was generated from 1000
bootstrap datasets in order to provide statistical support for each node.
4.3. qRT-PCR Analysis
cDNA was synthesised using the Invitrogen cDNA synthesis kit SuperScript IV,
according to manufacturer’s instructions. cDNA amplification was carried out using
SsoFast EvaGreen Supermix (BioRad), 250 nM of each gene-specific primer, and 1
µ
L of
1/25 diluted cDNA. Quantitative real-time PCR (qRT-PCR) reactions were performed
on the CFX96 Touch
™
Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).
Gene expression analysis was based on three biological and three technical replicates, and
was normalised with the reference gene CsaPP2AA3 [
71
]. Following an initial period of
3 min/95
◦
C, each cycle (40 in total) consisted of a denaturation step of 10 s at 95
◦
C and
annealing step of 10 s at specific primer temperature. Following the 40 cycles, a melting

Plants 2021,10, 1538 14 of 17
curve was obtained: the amplification products were heated in a gradient ranging from 65
to 95
◦
C in 5 s intervals. The primers used in the expression analysis are listed in Table S4.
4.4. Histological Analysis
For histological analysis, the flowers were collected and immediately fixed in 4% (w/v)
paraformaldehyde in 1
×
PBS, under vacuum infiltration, followed by overnight incubation
in the fixative solution at 4
◦
C. Samples were dehydrated, cleared, and embedded according
to the protocol described by Coen et al. [
72
]. Tissue sections with 8
µ
m thickness were
obtained using a microtome (SLEE) and mounted in pre-coated poly-L-lysine slides (VWR).
The sections were deparaffined and stained according to Viejo et al. [
73
], with modifications.
4.5. Yeast-2-Hybrid Analysis
Protein–protein interactions were analysed using a GAL4-based yeast hybrid system
(Matchmaker two-hybrid system; Clontech, Kusatsu, Shiga, Japan). Competent cells from
Saccharomyces cerevisiae strain AH-109 were transformed with pGBT9 (bait vector; Clontech)
and pGAD424 (prey plasmid; Clontech, Kusatsu, Shiga, Japan) derivatives, using the
LiAc/DNA/PEG transformation method. Self-activation assays and selection of positive
interactors were performed according to Causier and Davies [
74
]. To check for interaction,
double transformations were selected first in SD medium without leucine and tryptophan
and then in SD medium without leucine, tryptophan, and histidine supplemented with
3-aminotriazole. The primers used to construct the clones are listed in Table S4.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10
.3390/plants10081538/s1, Figure S1: GO functional annotation of the Castanea sativa de novo tran-
scriptome. Figure S2: Alignment of ABCDE-like amino acid sequences. Figure S3: Motif similarity
in B- and C- MADS-box proteins between C. sativa and A. thaliana,Q. suber and C. mollissima (or
S. lycopersicum in the case of TM6). Table S1: De novo assembly statistics of Castanea sativa transcrip-
tome before and after redundancy removal. Table S2: Castanea sativa transcriptome completeness as
determined by Benchmarking Universal Single-Copy Orthologous (BUSCO). Table S3: List of gene
accessions. Table S4: List of primers.
Author Contributions:
A.T.A. and R.S. designed and performed most of the experiments. M.R. and
M.J.N.R. contributed to the transcriptome assembly and bioinformatics analysis. S.A., L.M.-C. and
J.G.-L. contributed to the phenology characterisation. M.M.R.C. obtained funding, and designed and
supervised all the experiments. A.T.A. and R.S. wrote the manuscript, with contributions from all the
authors. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was funded by FCT/COMPETE/FEDER with the project grant POCI-01-0145-
FEDER-027980/PTDC/ASP-SIL/27980/2017—“FlowerCAST—Characterisation of genetic and envi-
ronmental determinants involved in reproductive development of Castanea sativa”. A.T.A. and S.A.
were supported by FCT with PhD grants (ref. SFRH/BD/136834/2018 and SFRH/BD/146660/2019,
respectively).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The generated de novo reads used in this study are openly available in
NCBI Sequence Read Archive under the accession number PRJNA744401.
Acknowledgments:
The authors would like to acknowledge the Galaxy Project for the freely available
resources that made the C. sativa transcriptome assembly possible. The authors are also grateful to
Isaura Castanheiro for allowing access to the chestnut orchard.
Conflicts of Interest: The authors declare no conflict of interest.
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